Evaluation of Six Protocols for Protein Extraction from Rice Young Panicles byTwo-Dimensional Electrophoresis

Abstract

ABSTRACT For most reported proteomics approaches, protein extraction and sample preparation are of crucial importance for optimal results. However, extraction of protein from crop plant tissues remains a great challenge due to low protein content and abundant secondary metabolites that prominently interfere with isoelectric focusing and subsequent proteomic analysis. Up until now, no attempts are focused on comparison of protein extraction procedures from rice young panicles. To establish a high-efficiency protein extraction protocol suitable for two-dimensional electrophoresis (2-DE) in rice young panicles, six protocols for protein preparation were evaluated: (1) Phenol extraction; (2) Mg/ Nonidet P-40 (NP-40) extraction; (3) Tris-Base/acetone precipitation; (4) SDS extraction; (5) trichloroacetic acid (TCA)/acetone/phenol extraction; (6) TCA/acetone precipitation. The study explicitly demonstrates that TCA/ acetone/phenol method provides a high-enhanced protein extraction efficacy from rice young panicles than other protocols in terms of the higher quality of 1-DE gel, the maximum number (450) of well-resolved protein spots, greater resolution and spot abundance. In addition, these methods also generated remarkably different twodimensional gel electrophoresis protein spot patterns. Twenty-nine of 30 visible differential extracted proteins were identified by MALDI-TOF-MS/MS analysis and were divided into eight categories according to molecular function. Accumulated data suggested that different extraction methods respectively necessitate certain special plant tissues due to different physicochemical properties of each protocol. Overall, this study, which is presented in this paper, will facilitate to providing a cornerstone of comparative proteomic analysis from rice young panicles, including other complicated plant tissues.